Multimodal Cleavable Reporters versus Conventional Labels for

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Multimodal Cleavable Reporters vs Conventional Labels for Optical Quantification of Accessible Amino and Carboxy Groups on Nano- and Microparticles Marko Moser, Nithiya Nirmalananthan, Thomas Behnke, Daniel Geißler, and Ute Resch-Genger Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b00666 • Publication Date (Web): 10 Apr 2018 Downloaded from http://pubs.acs.org on April 10, 2018

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Analytical Chemistry

Multimodal Cleavable Reporters vs Conventional Labels for Optical Quantification of Accessible Amino and Carboxy Groups on Nano- and Microparticles Marko Moser, 1,2,§ Nithiya Nirmalananthan,1,2,§ Thomas Behnke,1 Daniel Geißler,1 Ute ReschGenger1,* 1)

Federal Institute for Materials Research and Testing (BAM), Richard-Willstätter-Str. 11, D-12489 Berlin, Germany Institut fü r Chemie und Biochemie, Freie Universitä t Berlin, Takustrasse 3, D-14195 Berlin, Germany § Both authors contributed equally to this work. Corresponding Author* U.R.-G.: e-mail, [email protected]; phone, ++49(0)30-8104-1134; fax, ++49(0)30-810471134. KEYWORDS surface group quantification; polymer particle; cleavable linker; optical assay; fluorescent label 2)

ABSTRACT: Many applications of nanometer- and micrometer-sized particles include their surface functionalization with linkers, sensor molecules, and analyte recognition moieties like (bio)ligands. This requires knowledge of the chemical nature and number of surface groups accessible for subsequent coupling reactions. Particularly attractive for the quantification of these groups are spectrophotometric and fluorometric assays, which can be read out with simple instrumentation. In this respect, we present here a novel family of cleavable spectrophotometric and multimodal reporters for conjugatable amino and carboxyl surface groups on nano- and microparticles. This allows determination of particle-bound labels, unbound reporters in the supernatant, and reporters cleaved off from the particle surface as well as the remaining thiol groups on particle by spectrophotometry and inductively coupled optical emission spectrometry (32S ICP-OES). Comparison of the performance of these cleavable reporters with conductometry and conventional labels, utilizing changes in intensity and/or color of absorption and/or emission, underlines the analytical potential of this versatile concept which elegantly circumvents signal distortions by scattering and encoding dyes and enables straightforward validation by method comparison. The preparation and characterization of nanoparticles (NP) and microparticles (MP) and their use in the life and material sciences have been vivid research topics in the last decades.1-4 Such inorganic, organic, metal, or hybrid particles are employed, e.g., as carriers for functional molecules like dyes and drugs, reporters for signal enhancement strategies in optical assays, targeted probes in bioimaging studies, and nanoscale sensors.5-11 Applicationrelevant features of these particles include size (and size distribution) and shape, as well as optical, magnetic, catalytic or electrochemical properties used for signal generation. Of similar interest is their surface chemistry, which largely determines their colloidal stability, biocompatibility (e.g., formation of a protein corona12), and potential toxicity as well as further processing steps like the conjugation to biomolecules.13-18 The efficiency of surface functionalization is influenced by the applied conjugation strategy (e.g., direct labeling of surface groups compared to covalent attachment via linkers),5,17 the properties of the molecules to be bound like type of reactive group(s), bulkiness, and charge, and the characteristics of the particle surface.17-20 For example, tuning of particle hydrophilicity, e.g., by covalent attachment of polyethylene glycol (PEG)21 molecules and surface binding of target-specific biomolecules and ligands are

largely controlled by the chemical nature and accessibility of surface functionalities, particle morphology, and electrostatic attraction or repulsion.22,23 Many label-free and label-based methods have been used for the analysis and quantification of functional groups (FGs) on nanomaterials, that rely either on intrinsically present reporters or on differently sized labels, covalently bound to or electrostatically interacting with these FGs, for signal generation.4 Sophisticated analytical techniques such as nuclear magnetic resonance (NMR),16,24 X-ray photoelectron spectroscopy (XPS),25-27 as well as inductively coupled plasma optical emission spectroscopy (ICP-OES) or mass spectrometry (ICP-MS)28 can principally utilize intrinsic reporters and labels for FG determination – depending on the chemical nature of the respective FGs – but need expensive and complicated instrumentation and elaborated data analysis. Therefore, these methods are not really suitable for routine analysis. For process and quality control during particle manufacturing and surface functionalization, methods are desired that are fast, simple, yet selective and sensitive, and require only inexpensive instrumentation available in each laboratory. Ideal in this respect are electrochemical titration methods like conductometry or potentiometry, and optical methods. Electrochemical titration utilizes the smallest possible reporters, 1

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protons (H+) and hydroxide ions (OH-) for signal generation, and thus, yield the maximum number of accessible FGs. This maximum number typically equals the total number of (de)protonable surface functionalities, as demonstrated by us in a comparison of conductometry and solid state 13C-NMR for nano- and microparticles surface functionalized with polyacrylic acid.17 Electrochemical titration methods are inexpensive, but require a relatively large amount of sample and prolongated equilibration times.29-31 Moreover, they are sensitive to ionic contaminations originating e.g., from initiators of radical polymerization reactions, stabilizers, and salts. Optical methods like spectrophotometry and fluorometry require an absorptive or emissive label with a larger size compared to H+ and OH, like organic dyes covalently attached or adsorbed to the FGs of interest.16-18,23,26,32-34 Although this approach yields a smaller amount of FGs with a respective stoichiometry factor depending on label size, it provides important information on the number or density of accessible or derivatizable FGs required for subsequent (bio)conjugation reactions. This is essential for bioanalytical assays and other applications of such particles where surface functionalization or modification needs to be optimized regarding reaction conditions and minimization of the amount of precious and costly reagents (e.g., biomolecules).16-19,35 Optical assays, that can be performed with routine laboratory instrumentation involve the labeling with i.) conventional “always on” dyes (Scheme 1, panel i), the spectral features of which are not affected by conjugation, or ii.) dye precursors that become strongly absorptive or emissive upon reaction with the respective FG (“turn-on” or activatable labels) or color changing dyes. The latter show significant binding-induced shifts in their absorption and/or emission spectra (so-called “chameleon dyes”; Scheme 1, panel ii). This can enable spectral separation of reacted and unreacted labels and can thus omit tedious washing steps. In all cases, direct quantification of surfacebound dyes can be hampered by light scattering for particles with sizes exceeding about 50 nm or signal contributions from encoding dyes or the particle itself in the case of e.g., metal particles and semiconductor quantum dots. Also, the signal intensity of a surface-bound reporter can differ from that of the free label, particularly for fluorescence methods, as fluorescence is an environment-sensitive quantity and can be prone to quenching induced by dyedye interactions. This can render calibration tedious and erroneous. Aiming at simplifying optical surface group quantification and validation with complementary and independent analytical methods, we developed a platform of multimodal cleavable optical reporters for very common amino and carboxy functionalities (see Scheme 1, panel iii). These novel probes can be detected bound to the particle surface and after cleavage in solution spectrophotometrically as well as by ICP-OES or ICP-MS. In order to underline the potential of this versatile approach, we compared the results obtained with these cleavable probes with those derived from conventional dye labels as well as activatable and chameleon reporters. These measurements were complemented by conductometric titration, providing the

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total (or at least maximum) amount of FGs measurable with a reporter-based analytical method. Scheme 1. Dye-based Quantification/Labeling Strategies via Always ON, Activatable, and Cleavable Labels.

Materials and Methods Materials. Amino- and carboxy-functionalized, 100 nmand 1 µm-sized polystyrene particles (PSP) were purchased from Kisker Biotech GmbH (Germany). Prior to use, they underwent three washing, centrifugation (Centrifuge 5415D from Eppendorf (Germany), 40 min at 16000 g for 100 nm-sized PSP, 10 min at 5000 g for 1 µm-sized PSP), and resuspension (by ultrasonification) cycles. Ellman’s reagent (DNTB), propylamine, L-cysteine, N-Bocethylenediamine, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), N-sulfohydroxysuccinimide (sulfoNHS), N-hydroxysuccinimide (NHS) 6-aminofluorescein, NHS-fluorescein, O-(7-azabenzotriazol-1-yl)-N,N,N′,N′tetramethyluroniumhexafluorophosphate] (HATU), 4-morpholine-ethanesulfonic acid monohydrate (MES), tetrahydrofuran (THF), dichlormethane (DCM), dimethylsulfoxide (DMSO), dimethylformamide (DMF), methanol (MeOH), ethanol (EtOH), sodium dihydrogenphosphate, trifluoroacetic acid (TFA), triethylamine (TEA), sodium hydroxide, phosphoric acid and hydrochloric acid were purchased from Sigma Aldrich Co. (Germany), N-Succinimidyl-3-(2-pyridyldithio) propionate (SPDP, NHS-PDP), tris(2-carboxyethyl) phosphine hydrochloride (TCEP) were obtained from Thermo Fisher Scientific Germany BV & Co KG (Germany). All solvents were of spectroscopic grade. All solutions and buffers were prepared with Milli-Q water (Millipore). Synthesis of N-APPA. The carboxy-reactive cleavable reporter N-(aminoethyl)-3-(pyridin-2-yldisulfanyl)propanamide trifluoroacetate (N-APPA) was obtained by a slightly modified addition-elimination reaction from H. W. Chien et al.,36 of SPDP with a primary amine using N-Boc2


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ethylendiamine (see Supporting Information (SI), Figure S1). The latter inhibited cross reactions and facilitated purification by column chromatography on silica gel. The protection group was cleaved as reported by H. R. Krüger et al.37 For the synthesis of N-APPA, a solution of SPDP (50.38 mg, 161 µmol) dissolved in 6 mL DCM was added to a solution of N-Boc-ethylenediamine (64.83 mg, 403 µmol) and TEA (56 µL, 403 µmol) in 10 mL DCM. The reaction mixture was stirred for 1 h at room temperature (RT) and diluted with 5 mL DCM. The solvent was removed by vacuum evaporation and the solid sample was purified by column chromatography on silica gel using DCM: MeOH (20:1) and 1 % triethylamine as eluent. Boc-protected N-(aminomethyl)-3-(pyridin-2-yldisulfanyl)propanamide, obtained as light yellow solid, was then dissolved in 2 mL DCM followed by addition of 2 mL TFA (25 mM). The mixture was stirred over night at RT. DCM and TFA were removed by vacuum evaporation. The yellow oil was purified by column chromatography (reversed phase C18 43 g, water/methanol; linear gradient 0-100 %), yielding 55 mg (148 µM, yield 92 %) of the pure product, which was characterized by ESI-MS, 1H-NMR, and 13C-NMR (see SI, Figures S2-S5). Amino Group Quantification with NHS-Fluorescein. 20 µL of 100 nm- or 1 µm-sized aminated PSP (5 wt-%) in phosphate buffer (0.01 M, pH 8) were mixed with 2.075 µmol NHS-fluorescein, dissolved in 40 µL EtOH, and shaken at 500 rpm at RT for 16 h, followed by centrifugation at 16,000 g for 40 min for 100 nm-sized particles or at 5,000 g for 10 min for 1 µm-sized PSP, respectively. The supernatant was removed and the dye-labeled PSP suspension was washed three times with 300 µL phosphate buffer (0.01 M, pH 8) and twice with 100 µL EtOH. The amount of unreacted dye in the merged supernatants was quantified spectrophotometrically at λabs = 505 nm (ε = (72,000 ± 300) M-1cm-1) and fluorometrically (λexc = 485 nm, λem = 525 nm). Amino Group Quantification with Fluram. 0.175 µmol Fluram, dissolved in 50 µL DMSO, were added to 10 µL of 100 nm-sized aminated PSP (2.5 wt-%) or 1 µm-sized PSP (25 wt-%) in phosphate buffer (0.01 M, pH 8) and shaken at 300 rpm for 10 min at RT. 50 µL phosphate buffer were added and the mixture was shaken for 10 min. After adding 1900 µL THF for dissolving the PSP, the mixture was shaken for 10 min. The amount of reacted dye was quantified fluorometrically (λexc = 400 nm, λem = 482 nm). For the determination of the total amount of FGs, the PSP were dissolved in 1900 µL THF before adding 1.5 µmol Fluram. Amino Group Quantification with IR797. 0.175 µmol IR797, dissolved in 100 µL of a EtOH-DMSO mixture (10 Vol-% DMSO), were added to 10 µL of 100 nm-sized aminated PSP (2.5 wt-%), suspended in 100 µL basic ethanol, or to 110 µL of 1 µm-sized aminated PSP (5 wt-%) suspended in basic EtOH. The mixture was shaken at 500 rpm for 16 h at 50°C. The unreacted chlorinated dye in the supernatant was quantified spectrophotometrically at λabs = 803 nm (ε = (385,000 ± 2000) M-1cm-1) and fluorometrically (λexc = 750 nm, λem = 825 nm). Then, the PSP

were dissolved by adding 1190 µL THF and the reacted dye was quantified spectrophotometrically at λabs = 650 nm (ε = (148,000 ± 1000) M-1cm-1) and fluorometrically (λexc = 650 nm, λem = 695 nm). Amino Group Quantification with SPDP. 20 µL of aminated PSP (2.5 wt-%) were added to a solution of 2.1 µmol SPDP (for 100 nm-sized PSP) or 0.9 µmol (for 1 µm-sized PSP), dissolved in 30 µL DMSO and 50 µL phosphate buffer (0.01 M, pH 8), and shaken at 700 rpm for 45 min at RT, followed by centrifugation at 16,000 g for 40 min for 100 nm-sized particles and at 5000 g for 10 min for 1 µmsized PSP, respectively. The supernatant was removed and the PSP suspensions were washed twice with 350 µL phosphate buffer. The merged supernatant fractions containing unreacted, hydrolyzed SPDP (see SI Figure 11, left) were mixed with 100 µL of a TCEP stock solution (14 mM) in phosphate buffer to cleave the disulfide bond of SPDP. The reaction mixture was shaken at 700 rpm for 45 min at RT. The amount of 2-thiopyridone (2-TP) formed was quantified spectrophotometrically at λabs = 343 nm (ε = (8000 ± 100) M-1cm-1). Additionally, 100 µL of a TCEP stock solution were added to the washed PSP (see SI Figure 11, right), resuspended in 500 µL phosphate buffer. After shaking at 600 rpm for 45 min at RT, the PSP suspension was washed twice with phosphate buffer. The amount of 2-TP in the merged supernatant fractions was determined spectrophotometrically. The thiol groups formed at the particle surface were quantified by the Ellman’s assay and ICP-OES. Activation of Carboxy Groups on PSP. 120 µL EDC (150 mM) and 60 µL sulfo-NHS (300 mM) in MES buffer (0.05 M; pH 5) or 60 µL HATU (300 mM) in DMF were added to 100 µL of 100 nm- or 1 µm-sized carboxylated PSP (5 wt-%) in MES buffer. The mixture was shaken at 600 rpm for 1 h at RT, followed by one washing step using 400 µL phosphate buffer (0.01 M, pH 8). To avoid sulfur contamination for subsequent ICP-OES validation, the activation procedure was slightly modified. Instead of MES buffer, phosphate buffer (0.01 M, pH 5.5) was used and sulfo-NHS was replaced by NHS. Carboxy Group Quantification with 6-Aminofluorescein. 520 µL of the activated carboxylated PSP suspension (0.96 wt-% for both 100 nm- and 1 µm-sized PSP) were added to a solution of 1.750 µmol 6-aminofluorescein (for 100 nm-sized PSP) or 0.25 µmol (for 1 µm-sized PSP), dissolved in 80 µL DMSO. The reaction mixture was shaken at 600 rpm for 16 h at RT, followed by centrifugation at 16,000 g for 40 min for 100 nm-sized particles or at 5,000 g for 10 min for 1 µm-sized PSP, respectively. The supernatant was removed and the dye labeled PSP suspension was washed three times with 500 µL phosphate buffer (0.01 M, pH 8). The amount of unreacted dye in the merged supernatants was then quantified spectrophotometrically at λabs = 489 nm (ε = (76,000 ± 500) M-1 cm-1) and fluorometrically (λexc = 480 nm, λem = 505 nm). Carboxy Group Quantification with N-APPA. 580 µL of the activated carboxylated PSP suspension (0.86 wt-% for both 100 nm- and 1 µm-sized PSP) were added to a 3



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Scheme 2. Overview of Applied Optical Assays and Reporters Used.a

aAbsorption spectra of applied reagent (black), absorption spectra of reporter (green), emission spectra of applied reagent (blue), and emission spectra of reporter (red). A larger presentation of the spectra can be found in Figure S17 in the Supporting Information.

solution of 1.750 µmol N-APPA (for 100 nm-sized PSP) or 0.25 µmol (for 1 µm-sized PSP), dissolved in 80 µL methanol. The reaction mixture was shaken at 600 rpm for 16 h at RT, followed by centrifugation at 16,000 g for 40 min for 100 nm-sized particles and at 5000 g for 10 min for 1 µmsized PSP, respectively. The supernatant was removed and the labeled PSP suspension was washed three times with 500 µL phosphate buffer (0.01 M, pH 8). 20 µL of a TCEP stock solution (40 mM) in phosphate buffer was added to the merged supernatant fractions with unreacted N-APPA (see SI, Figure 13, left) and shaken at 600 rpm for 45 min at RT. The amount of 2-TP was quantified spectrophotometrically at λabs = 343 nm (ε = (8000 ± 100) M-1cm-1). Additionally, 50 µL of a TCEP stock solution were added to the washed PSP (see SI Figure 13, right), which were resuspended in 350 µL phosphate buffer. After shaking at 600 rpm for 45 min at RT, the PSP suspension was washed twice with phosphate buffer. The amount of 2-TP in the merged supernatant fractions was determined spectrophotometrically. The thiol groups formed at the particle surface were quantified with the Ellman’s assay and ICP-OES as described for amino group quantification with SPDP. Ellman’s assay.32 Ellman’s reagent was used as a 3.4 mM solution in phosphate buffer (0.01 mM, pH 8). Thiol containing PSP samples were mixed with 50 µL of the Ellman`s stock solution. After a reaction time of 4 h, the PSP were removed by centrifugation. The supernatant was diluted to a volume of 3 mL with phosphate buffer in (10 x 10) mm quartz cuvettes. The thiol concentration was then determined from the absorbance of 2-nitro-5-thiobenzoic acid (NTB2-) at 409 nm, using the previously32 determined molar absorption coefficient (ε = (14,100 ± 200) M-1cm-1).

Molar absorption coefficients. The molar decadic absorption coefficients (ε) of all dyes were determined by measuring the absorbances of a concentration series of each dye (absorbance < 1) in the respective solvent/solvent mixture. The slopes of the concentrationdependent absorbance values at the dye´s absorption maxima were fitted with a linear function according to the Beer-Lambert law. Instrumentation. Absorption Measurements. Absorption spectra were measured with the calibrated double beam spectrophotometer Specord 210plus from Analytik Jena (Germany) at RT in (10 x 10) mm quartz cuvettes from Hellma GmbH (Germany). Fluorescence Measurements. Fluorescence emission spectra of solutions were measured with a Microplate Reader Infinity M200 Pro from Tecan Inc. (Austria) at RT in microtiter plates from Fischer Scientific GmbH (Germany) or with the fluorometer Fluoromax4 from Horiba Scientific (Germany). Photoluminescence quantum yields (Φ). Φ values, providing the ratio of the number of emitted photons and absorbed photons, were determined absolutely with an integrating sphere setup from Hamamatsu (Quantaurus-QY C11347-11) previously evaluated by us.38 All Φ measure ments were performed at 25°C using 10 mm x 10 mm long neck quartz cuvettes from Hamamatsu. ICP-OES Measurements. Quantitative ICP-OES analysis of the sulfur concentration of the suspended PSP was performed with a 5110 ICP-OES from Agilent Technologies. 32S was detected at 180.669 nm. Conductometric Titration. Conductivity measurements providing the maximum or termed here total number of 4


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Table 1. Quantification of Total and Accessible Amount of Surface Functional Groups. Data given as absolute numbers and relatively compared to the total amount. Particle dia- Conductometry a meter [nm] [µmol/g NP]

Aminated PSP

Activatable and chromogenic reporter [µmol/g NP]

Conventional dye [µmol/g NP]

Cleavable reporter [µmol/g NP]

NHS-fluorescein

Fluram

IR 797

SPDP

100

1027 ± 44 (100%)

150 ± 30 (14.6%)

130 ± 7 (12.7%)

95 ± 9 (9.3%)

130 ± 6 (12.7%)

1000

156 ± 31 (100%)

5.59 ± 1.04 (3.6%)

3.85 ± 0.85(2.5%)

3.57 ± 1.01 (2.3%)

6.03 ± 0.31 (3.9%)

6-Aminofluorescein

N-APPA

100

372 ± 58 (100%)

60 ± 25 (16.1%)

-

-

59 ± 6 (15.9%)

1000

224 ± 21 (100%)

10 ± 3.10 (4.5%)

-

-

9.52 ± 0.51 (4.3%)

Carboxy PSP

a

Determination of total amount under argon atmosphere.

(de)protonable FGs were done with a Modul 856 conductometer (Methrom). For complete protonation/deprotonation of the FGs on the PSP surface (SI, Figure S6), the conductivity of the PSP suspensions was adjusted to 100 μS/cm with either HCl or NaOH prior to FG titration. The samples contained 10 mg of PSP in 80 mL Milli-Q water, were treated, i.e., titrated with base (10 mM NaOH) or acid (10 mM HCl) in 20 µL steps, under argon atmosphere, thereby excluding CO2. All relative standard deviations of the optical assays were derived from measurements of six independent samples. In the case of the ICP-OES measurements, the relative standard deviations were obtained from three independent measurements.

cally and fluorometrically after particle removal (Scheme 2, panel i). For complete reaction of all accessible amino surface groups, an excess of NHS-fluorescein is mandatory. Here, at least a 4 fold excess of NHS-fluorescein was necessary to achieve quantitative FG labeling (see SI, Figure S7). With this method, the amount of accessible amino groups on the 100 nm- and 1 µm-sized aminated PSP was determined to 14.6 % and 3.6 % of the total amino groups, respectively.

Results and discussion Aiming at the development of methods for the optical determination of accessible surface groups on nanomaterials, microparticles, and planar substrates, we assessed different types of optical labels (i.e., NHS-fluorescein, 6-aminofluorescein, Fluram39,40, IR 79741-43) and novel cleavable reporters (i.e., SPDP, N-APPA) summarized in Schemes 1 and 2. As a prerequisite for this reporter comparison, the total amount of FGs on representatively used 100 nm- and 1 µm-sized aminated or carboxylated PSP was determined conductometrically. The results, obtained with a protocol validated by us for the determination of the total amount of carboxy groups on differently sized and surface modified PSP by comparison with NMR spectroscopy,16 are summarized in Table 1. For aminated particles, validation was done with the Fluram assay and dissolved nanomaterials (cf. Figure 1 for 100 nm-sized PSP: 1010 ± 25 µmol/g and cf. SI, Figure S14 for 1 µm-sized PSP: 145 ± 15 µmol/g). The total amount of FGs was subsequently used for calculating the ratios of the accessible amount of FGs given in Table 1. Amino Group Quantification with NHS-Fluorescein. Direct quantification of the amount of amino groups on differently sized PSP with this label was hampered by scattering and self-quenching of particle-bound dyes. Consequently, the amount of amino groups was calculated as difference between the amount of initially applied dye and the amount of unreacted reporter in the supernatant (see Scheme 1). The latter was determined spectrophotometri-

Figure 1. Method comparison for amino (top) and carboxy (bottom) group quantification on 100 nm PSP. (a) Particle dissolution is required for on surface quantification. The corresponding results for the 1 µm aminated and carboxylated particles are shown in the SI (Figures S15 and S16).

Amino Group Quantification with Fluram. The colorless dye precursor Fluram (see Scheme 2, panel ii) forms a yellow product with primary amines, that displays a strong emission between 400 and 700 nm. A mechanism for this reaction, which is astonishingly not yet fully understood, is 5



suggested in the SI (Figure S8). Since a direct quantification of particle-bound Fluram is hampered by light scattering and hydrolysis of unreacted Fluram in the supernatant yields a non-emissive product, this method is restricted to particle systems which can be easily dissolved. Then, particle-bound Fluram can be quantified optically without interferences. We dissolved here Fluram-modified PSP in THF. Our results also revealed that the spectral position of the absorption and emission bands of the reaction product of Fluram with primary amines depends to a certain extent on the substitution pattern of the amine analyte, since the amine nitrogen atom becomes part of the chromophore π electron system. This follows, e.g., from the differences in the exemplarily shown absorption and emission spectra of the Fluram products of L-cysteine and propylamine in Figure 2.

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ously performed studies with its water soluble analog IR783, bearing two sulfonic acid groups (not shown here), required long reaction times (up to 3 days!) and gave only poor reaction yields in aqueous media. The reaction of the chameleon dye IR797 with primary amines (see SI, Figure S9) is accompanied by strong blue shifts in absorption and emission (see Schema 2, panel ii). The sizes of the spectral shift as well as ε and Φ are considerably influenced by the amine analyte substituting the chloro group of IR797. This is exemplarily underlined by a comparison of the spectroscopic properties of the reaction products with propylamine (ε650nm ≈ 156,000 M-1cm-1, Φ = 0.35), aniline (ε702nm ≈ 54,000 M-1cm-1, Φ = 0.26), and PEG-amine (ε652nm ≈ 109,000 M-1cm-1 Φ = 0.31) (see Figure 3). This makes an

Figure 3. Absorption (solid lines) and emission (dashed lines) spectra of IR 797 conjugates with propylamine, PEG-amine, aniline, and aminated PSP. Inset: Calibration curves of IR 797 with propylamine, PEG-amine, and aniline.

Figure 2. Absorption (solid lines) and emission (dashed lines) spectra of Fluram conjugates with propylamine, L-cysteine, and aminated PSP. Inset: Calibration curves of Fluram obtained with propylamine.

amine compound-specific calibration essential for an accurate quantification. Here, propylamine was used for calibration (see SI, Figure S10) as the absorption and emission spectrum of its reaction product with IR797 excellently match with that of the reaction product of IR797 and aminated PSP. In order to quantify surface-bound dye molecules and to obtain a mass balance, the dye-functionalized PSP were dissolved in THF and subsequently measured. Attempts to spectroscopically quantify the unreacted chlorinated dye in the supernatant after assuring a complete reaction with amino surface groups failed as it decomposed rapidly in THF. With this method, the amount of accessible amino groups on 100 nm- and 1 µm-sized aminated PSP was obtained to 9.3 % and 2.3 % of the total FG, respectively (see Table 1). Amino Group Quantification with SPDP. Prior to surface FG analysis, reductive cleavage of the SPDP reporter with TCEP was assessed spectrophotometrically and with 32S ICP-OES. This gave an average thiol recovery of at least 95 %, underlining the quantitative nature of this reaction. This is mandatory for FG quantification. Subsequently, SPDP was used for amino group quantification on differently sized PSP, thereby analyzing the unbound SPDP and indirectly the surface-bound SPDP after cleaving of 2-TP from the PSP surface by addition of TCEP

This can also affect ε and Φ of the measured Fluram product (see Figure 2; for reaction with propylamine: ε385nm ≈ 4000 M-1cm-1 and Φ = 0.26, for reaction with L-cysteine: ε345nm ≈ 3800 M-1cm-1 and Φ = 0.21). Hence, the choice of a suitable model system for correlating the absorption or fluorescence signal to analyte concentration is really relevant for an accurate analyte quantification. For the fluorometric quantification of amino groups on PSP, propylamine was used as a model system. Its suitability is underlined by the good match of the absorption and emission spectra of the Fluram products resulting for aminated PSP and propylamine. Hence, its use for calibrating the absorption and emission measurements (see inset in Figure 2) is expected to properly consider the compound-specific ε and Φ of the Fluram reaction product with aminated PSP. With this method, the amount of accessible amino groups was determined to 12.7 % and 2.5 % of the total FG for 100 nmand 1 µm-sized aminated PSP, respectively (see Table 1). Amino Group Quantification with IR797. The hydrophobicity of the chameleon dye IR797 led to dye precipitation in aqueous solutions, hence, this method was performed in EtOH. This dye was chosen despite its hydrophobicity as it provides relatively short reaction times and reasonably high reaction yields in EtOH. In contrast, previ6


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(see SI, Figure S11). The amount of 2-TP cleaved off from the PSP surface equals the amount of amino groups accessible for the SPDP reporter. We determined 12.7 % and 3.9 % of the total FG for 100 nm- and 1 µm-sized aminated PSP, respectively (see Table 1) via cleaved 2-TP. These results were confirmed by determining the difference of the amount of applied SPDP and unreacted hydrolyzed SPDP in the supernatant (see Figure 1). The latter yielded higher standard deviations. Release of 2-TP from the particle surface leads to formation of thiol surface groups (see Figure 4). These groups were subsequently quantified with 32S ICP-OES and the Ellman’s assay, following a previously reported protocol.32 The excellent match of these results with those derived from the SPDP method additionally confirmed the reliability of our cleavable probe concept.

ments addressing possible dye adsorption, the amount of dye molecules was also determined analogously without activation. This clearly revealed the absence of dye adsorption. With this indirect method, the amount of accessible carboxy groups on 100 nm and 1 µm carboxy PSP was determined to 16.1 % and 4.5 % of the total FGs, respectively. Carboxy Group Quantification with N-APPA. In order to expand the strategy of cleavable reporters to carboxy groups, we synthesized the cleavable reporter N-APPA. N-APPA was then coupled to activated carboxylated PSP via its primary amino group; the reaction mechanism is shown in the SI (Figure S13). The 2-TP reporter, formed upon reductive cleavage of the disulfide bond with TCEP, was used for quantifying the amount of unreacted N-APPA in the supernatant. Additionally, PSP-bound N-APPA was reductively cleaved yielding the 2-TP reporter and thiolfunctionalized PSP, which could be then utilized for method validation via mass balances, Ellman’s assay, and 32S ICP-OES. These measurements yielded 15.9 % and 4.3 % of accessible carboxy groups (see Table 1) for 100 nm and 1 µm PSP. The results from the spectrophotometric detection of 2-TP are in good agreement with those of the Ellman’ assay and 32S ICP-OES (see SI, Figure S14). Density of FGs. The results obtained with the cleavable reporters were subsequently utilized to calculate the density of accessible FGs. Accessible in this context implies accessible for the conjugation to these cleavable reporters, as this number could be different (i.e., smaller) for larger biomolecules. For the calculation of the FG densities, monodisperse spherical PS particles with a smooth surface were assumed. As expected for the outmost layers, we obtained FG densities of 1.4 FG/nm2 for the 100 nm and 0.6 FG/nm2 for the 1 µm aminated PSP, as well as 0.6 FG/nm2 for the 100 nm and 1.0 FG/nm2 for the 1 µm carboxylated PSP, respectively. Here, it also needs to be considered that in general, there is a lower and an upper limit for the quantification of FGs on particle surfaces. For a very low FG density, quantification is limited by the limit of detection (LOD) of the respective detection method used, e.g., conductometry, spectrophotometry, or fluorimetry. We determined the LOD for each quantification method from calibration curves using either dilution series of small organic molecules containing the respective functional groups (for conductometry, and the activatable/ chromogenic dye reporters), or from dilution series of the reporter dyes itself (for the common fluorescence dyes and the cleavable reporters). However, one can simply increase the amount of sample and thus, the amount of FGs to obtain measurable values. The closeness of the match of the amount of FGs obtained with differently sized surface conjugated reporters and conductometric measurements under these conditions depends particularly on the occurrence and extent of sterical hindrances on the labeling reactions. In contrast, if the FG density is very high, the fraction of accessible FGs will most likely decrease, as the extent of sterical hindrance between the FGs increases and reporter size will start to matter, thereby

Figure 4. Validation of surface group analysis with SPDP using ICP-OES and Ellman’s assay exemplarily for 100 nm-sized aminated PSP.

Carboxy Group Quantification with 6-Aminofluorescein. In this case, the amount of accessible functionalities was determined spectrophotometrically and fluorometrically from the difference between the amounts of initially applied and unreacted 6-aminofluorescein in the supernatant (indirect quantification), respectively, see Scheme 2 (panel i). Prior to surface group analysis, the conjugation reaction was optimized using the common activation reagents EDC/sulfo-NHS and HATU. Thereby, a slightly higher coupling efficiency was observed for HATU (one-step-activation) compared to two step EDC/sulfoNHS activation. In order to avoid underestimation of the amount of carboxy groups, the carboxylated PSP were reacted with increasing amounts of dye for both carboxy activation methods. This concentration series revealed that at least a 5-fold excess of dye is necessary for quantitative FG labeling (see SI, Figure S12). In control experi7



introducing larger deviations between different labels and hence detection methods.

tion schemes as shown here exemplarily for the chosen disulfide linker. This linker introduces quantitatively new surface groups, here thiol groups, which can be subsequently quantified with ICP-OES and the Ellman’s assay. Moreover, this versatile concept circumvents distortion of optical signals by particle scattering and reporter-reporter interactions. In order to expand this concept to other optical methods, we are currently evaluating cleavable reporters with fluorescent groups and fluorine-containing reporter parts, which allow for surface group quantification with optical assays, elemental analysis, ICP-OES, NMR, XPS, and reference-free total reflection X-ray fluorescence (TXRF).44 For the reporters and particle systems used here, only small differences in the resulting number of accessible surface groups are obtained despite the slightly different label sizes, which seem to be more pronounced for the nanoparticles as to be expected. A plausible reason could be the synthesis of aminated particles by surface modification of carboxylated particles, e.g., with triamines. This automatically introduces a linker between the particle surface and the functional group. Possible effects of reporter size will be addressed in the near future for selfmade polymer particles with systematically varied numbers of surface groups. S17 Absorption and emission spectra of applied reagents.

Conclusion and Outlook In summary, with SPDP and N-APPA, we developed a novel family of cleavable reporters for the quantification of accessible amino and carboxyl surface groups on nano- and microparticles. These cleavable labels, which are easily synthesized in high yields, allow for the quantification of particle-bound reporters, unbound reporters in the supernatant, and reporters cleaved off from the particle surface. These labels can be also indirectly quantified via the formation of new functional groups on the particles that could be measured with ICP-OES and the Ellman´s assay, an already evaluated optical assay for thiol groups. The advantages of these comparably small, versatile and multimodal reporters were demonstrated by comparison with conventional fluorescent labels, here the dyes NHS-fluorescein and 6-aminofluorescein as well as with the activatable “turn-on” reporter Fluram and the chameleon dye IR797. Although the conventional fluorescein derivatives are spectrophotometrically and fluorometrically detectable, they could only be quantified indirectly via the amount of unreacted dyes in the particle free supernatant to prevent signal distortions by particle scattering and selfquenching of particle-bound dyes (cf. Table 2). This applies also for the activatable dye Fluram and the chameleon dye IR797. As Fluram is unstable in aqueous solutions, the amount of surface-bound dyes could only be determined after particle dissolution in an organic solvent. IR797, enabled the spectroscopic determination of unreacted dye and accessible groups of occasionally dissolved particles. Moreover, application of both Fluram and IR797 requires calibration with model systems with spectroscopic features closely matching those of the reaction products to be quantified, to account for target-specific changes in ε and Φ. Table 2. Applicability of the Applied Methods. Conductometry Total amount Accessible amount Unbound On surface Cleaved from surface Mass balance Spectrophotometry Fluorometry a. c.

+ – – + – – – –

[i] Convent. dye

– + + – – – + +

[ii] Precursor

+a + –/+b + – – + +

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ASSOCIATED CONTENT Supporting Information Supporting Information Available: S1-S5. Synthesis and Analytical Characterization of N-APPA. S6 Conductometry. S7-S11 Amino Group Quantification. S12-S13 Carboxy Group Quantification. S14-S15 Method validation and comparison. The Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION

[iii] Cleavable

Corresponding Author * U.R.-G.: e-mail, [email protected]; phone, ++49(0)30 8104-1134; fax, ++49(0)30-8104-71134.

– + + –/+c + + + –

ACKNOWLEDGMENT This project has received funding from the EMPIR programme (project 14IND12 Innanopart) co-financed by the Participating States, from the European Union’s Horizon 2020 research and innovation programme. We gratefully acknowledge financial support from the Federal Ministry for Economic Affairs and Energy (BMWi-10/12). We would like to thank Johannes Horst Budau for graphic elaboration of our scheme ideas and Christopher Kläber for technical assistance.

after previous particle dissolution, b. possible for chameleon dyes, detectable with complementing methods, e. g., mass spectroscopy

This comparison underlines the general advantages of our rationally designed cleavable reporters SPDP and N-APPA for the quantification of functional groups on particle surfaces. Firstly, a mass balance can be obtained from measurements of the unreacted reporter and the cleaved off reporter. Secondly, the choice of a suitable cleavable reporter can enable a straightforward and simple validation with other analytical methods relying on different detec-

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Multimodal Cleavable Reporters versus Conventional Labels for

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